PHARMACOLOGIA
RESEARCH ARTICLE
DOI: 10.5567/pharmacologia.2015.360.370
Modulation of Arachidonic Acid Metabolism in the Rat Kidney by
Resveratrol: Implications for Regulation of Blood Pressure
1
1
Fawzy A. Elbarbry, 2Anke Vermehren-Schmaedick and 1Deepa Rao
School of Pharmacy, Pacific University, Hillsboro, OR ,USA
Department of Physiology and Pharmacology, Oregon Health and Science University, Portland
2
ABSTRACT
Background and Objective: The objective of this study was to investigate the effects of resveratrol (RES) on
Arachidonic acid (AA) metabolism in the kidney and its effect on arterial blood pressure, using spontaneously
hypertensive rats (SHR) as a model system. Methods: Rats were exposed to either drinking water alone (control) or
RES (20 or 40 mg kgG1) added to drinking water for 7 weeks. Mean Arterial Pressure (MAP) was measured at
7-day intervals throughout the study. At the end of treatment, rats were euthanized, followed by preparation of kidney
microsomes to measure enzymes involved in regulation of vasoactive metabolites: CYP4A, the key enzyme in the
formation of 20-hydroxyeicosatetraenoic acid and the soluble epoxide hydrolase, which is responsible for the
degradation of the vasodilator metabolites such as epoxyeicosatetraenoic acids. Effect of RES on kidney expression of
CYP4A was also investigated by immunoblotting. Results: Treatment with RES resisted the progressive rise in MAP
in the developing SHR in a dose-dependent manner. Consistent with these data, RES treatment led to significant
reductions in both, the expression and activity of renal CYP4A isozymes, as well as the activity of soluble epoxide
hydrolase (sEH). Conclusion: The presented studies show that RES modulates the metabolism of AA by both P450
enzymes and sEH in SHR rats, which may represent a novel mechanism by which RES protects SHR rats against the
progressive rise in blood pressure.
Key words: Resveratrol, spontaneously hypertensive rats, arachidonic acid, metabolism, soluble epoxide hydrolase,
hypertension
Pharmacologia 6 (8): 360-370, 2015
sodium transport2-4. Renal Cytochrome P450
(CYP)-mediated metabolism of AA produce either
hydroxyeicosatetraenoic acids (HETEs; particularly,
19- and 20-HETE) or epoxyeicosatetraenoic acids
(EETs, Fig. 1)2, 3, 5, 6.
In the renal and peripheral vasculature, 20-HETE
is a potent vasoconstrictor and is involved in
tubule-glomerular feedback and the autoregulation of
renal blood flow and glomerular filtration rate4,7.
Additionally, several studies have demonstrated that the
vasoconstriction effect of angiotensin II and nor
epinephrine is linked in to the endogenous formation of
20-HETE in renal vascular smooth muscle and
blockade of the Kca channels8,9. Moreover, infusion of
inhibitors of the 20-HETE formation into the renal
artery was found to attenuate the vasoconstrictor effect
of these agents7,10. CYP4A11 and CYP4F2 were found to
be responsible for the production of 20-HETE in the
human kidney2 while CYP4A1 was found to exhibit the
highest catalytic activity for the formation of 20-HETE
INTRODUCTION
Hypertension is a major predictor for fatal and nonfatal cardiovascular diseases. According to the Centers
for Disease Control and Prevention (CDC), it was
estimated that hypertension affects more than 30% of
the adults in the United States (http://www. cdc.gov/
blood pressure/facts.htm). Although, there is still
uncertainty about the pathophysiology of hypertension,
many interrelated factors have been found to contribute
to persistent blood pressure elevation1. Common causes
of hypertension include the following: (1) Vascular
resistance, (2) Oxidative stress, (3) Endothelial
dysfunction and (4) Salt sensitivity, among others.
Several studies have indicated that Arachidonic Acid
(AA) metabolites play a critical role in the regulation of
renal vascular tone, tubuloglomerular feedback and
Corresponding Author: Fawzy A. Elbarbry, School of Pharmacy,
Pacific University, 222 SE 8th Ave., Hillsboro, OR 97123, USA
Tel: 503-352-7356 Fax: 503-352-7270
© 2015 Science Reuters, UK
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PHARMACOLOGIA
RESEARCH ARTICLE
CO2H
Arachidonic acid
CO2H
o
CO2H
CYP
4A
CYP
2C
EETs
OH
HETEs
Vasodilatation
Vasoconstriction
Resveratrol (RES)
Epoxide
Hydrolase
CO2H
OH OH
DHETEs
Inactive
Fig. 1: Metabolism of arachidonic acid by CYP enzymes and epoxide hydrolase. The sign “x” indicates the proposed
sites for the anti-hypertensive effect of RES
in the rat kidney, followed by CYP4A2 and CYP4A311.
Overwhelming evidence has established that the phase
of rapid elevation of blood pressure in the spontaneously
hypertensive rat (SHR) is associated with increased
production of 20-HETE by CYP4A isoforms expressed
in the renal tubules and blood vessels3. Conversely,
selective inhibition of CYP4A enzymes resulted in acute
reduction in blood pressure in SHR12.
In contrast to 20-HETEs, EETs, produced by the
endothelium, are potent vasodilators and considered as
antihypertensive13. Several studies have led to the
proposal that EETs serve as the Endothelium-Derived
Hyperpolarizing Factor (EDHF)13. Deficit in EETs,
especially 5,6-EET, was found to render the rat sensitive
to salt-induced elevations of blood pressure14. While
CYP2C8/9/18/19 are the predominant isoforms
responsible for the metabolism of arachidonic acid to
biologically-active EETs, CYP2C11 and CYP2C23 are
responsible for most of the renal epoxygenase activity in
rats3. Conversion of the biologically active EETs to the
inactive metabolites, dihydroxyeicosatetraenoic acids
(DHETEs), by soluble epoxide hydrolase (sEH) has
been reported in the SHR3 (Fig. 1). Consistent with the
latter findings, treatment of SHRs with inhibitors of
sEH lowers blood pressure3,15. Therefore, modulation
of AA hydroxylase activity (i.e., 20-HETE and
EETs-forming activity) could potentially have a
significant impact on the development and progression
© 2015 Science Reuters, UK
of hypertension. Such modulation can occur in response
to administration of drugs, herbal remedies, or even
dietary supplements.
The idea of modulation of drug-metabolizing
enzymes as a principal means by which dietary
constituents affect the risk of disease was first
outlined by Wattenberg16 and further elaborated by
Prochaska et al.17. Resveratrol (RES) is a natural
phytochemical which is a phytoalexin commonly found
in grapes, berries and peanuts. Several recent studies
have shown that RES prevents or slows the progression
of a wide variety of illnesses, including cancer,
cardiovascular disease and ischemic injuries, as well as
enhances stress resistance and extend the life spans of
various organisms from yeast to vertebrates18. RES has
been shown to have vasorelaxant effects through the
stimulation of Ca2+ activated K+ channels and enhancing
the nitric oxide signaling in the endothelium19,20. It has
also been shown that the treatment of SHR with RES
attenuated the hypertension through the modulation
of nitric oxide synthase20. In addition, a recent
meta-analysis on 247 patients by Liu et al.21 on the effect
of RES on blood pressure indicated that RES
consumption significantly decreases systolic blood
pressure at high doses ($150 mg dayG1)21. While the data
on the effects of RES on high blood pressure have been
validated in vitro and in vivo, the mechanisms responsible
for the effects remain to be fully elucidated. The current
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RESEARCH ARTICLE
study was undertaken to examine the anti-hypertensive
effect of RES in a hypertensive rat model and to
investigate its effect on AA-metabolizing enzymes in the
kidney.
concentrations provided approximately 20 and
40 mg kgG1 RES, respectively. The final ethanol
concentrations in the low dose and high dose group were
0.015 and 0.03%v/v, respectively. The amount of ethanol
present in the solutions was well below the oral LD50
concentration of 10.6 g kgG1 in rats23.
RES stability in the Pluronic® F127 formulations
was assessed using UV absorbance on a Biotek Sunergy
2 microplate reader at 306 nm. Briefly, RES stock
solutions in ethanol were prepared at 15 mg mLG1 or
25 mg mLG1 and diluted into water, 1, 2.5 or 5%
Pluronic® F127 solutions for a final concentration of
RES at 0.15 or 0.25 mg mLG1, respectively. The samples
were stored at room temperature. Blank solutions of
water, 1, 2.5 and 5% Pluronic F127 solutions were
prepared to account for vehicle effects. Samples were
assessed on day 1 (day of preparation) and day 3
following a 1:100 dilution in their respective vehicles.
Absorbance was recorded at 306 nm. By day 4, samples
showed precipitation of RES and no additional
measurements were made. Based on the presented data
the RES at low and high doses showed the highest
stability in 5% Pluronic® F127 solution.
MATERIALS AND METHODS
Materials: Resveratrol was purchased from TCI
(Portland, OR). Lutrol F127 Pluronic® (F127) was
kindly donated by BASF (Florham Park, NJ). Ethanol
USP was purchased from VWR (Radnor, PA).
Arachidonic acid and 20-HETE were purchased
from Cayman Chemical Company (Ann Arbor, MI).
Sprague-Dawley rat kidney microsomes for in vitro
studies were purchased from BD Biosciences (Woburn,
MA). NADPH and all reagents used for microsomal
preparation, determination of protein content and
enzyme assays were purchased from Sigma-Aldrich
(ST. Louis, MO). CYP4A1/2/3 polyclonal antibody
was purchased from Life Span Biosciences, Inc.
(Seattle, WA). Acetonitrile and methanol were of High
Pressure Liquid Chromatography (HPLC) grade and
obtained from Fisher Scientific (Pittsburg, PA). All other
chemicals used were of analytical grade VWR
(Radnor, PA).
Resveratrol(RES) treatment: Following acclimation
in the laboratory, animals were randomly divided into 3
groups of 5 animals each. Group one served as the
control group, whereas groups two and three were
provided with resveratrol in the drinking water at
concentrations of 20 and 40 mg kgG1, respectively, for
7 weeks. Fresh RES solutions were made from stock
twice a week. The selection of RES doses was based on
preliminary studies and previous studies21,23.
Animals: Male 14-week-old Spontaneously
Hypertensive Rats (SHR) were obtained from Harlan
Laboratories (Madison, WI). All animals were
maintained under controlled housing conditions of light
(6 am-6 pm) and temperature (22°C) and received
standard laboratory chow and water ad libitum. All rats
were allowed at least 2 weeks to become acclimated to
the housing conditions and ensure steady and reliable
blood pressure readings before use in experiments. All
procedures were approved by the Institutional Animal
Care and Use Committee of Pacific University and
Oregon Health and Science University.
Mean Arterial Pressure (MAP) measurements:
Blood pressure was measured as previously described by
Vermehren-Schmaedick et al.24. All blood pressure
measurements were performed in conscious animals.
For each SHR rat, blood pressure was measured
once every week using a non-invasive method based
on determining the tail blood volume with a
volume-pressure recording sensor and an occlusion
tail-cuff (CODA system; Kent Scientific, Torrington,
CT). Previously, it have been indicated that blood
pressure values obtained with the non-invasive tail-cuff
method are not significantly different from the values
obtained in the same animals by direct measurements in
the femoral artery24. Blood pressure was measured by
multiple readings in individual rats, until an average of
15 stable measurements was obtained. Results are shown
as the average of MAP values obtained from individual
rats.
Preparation of Resveratrol (RES) solutions: RES
stock solutions were prepared in pure ethanol at a
concentration of 3% w/v. Lutrol F127 Pluronic®, a
GRAS excipient recognized by the FDA was prepared at
5% w/v in DI water to keep the RES dissolved in the
solution for the duration of the study. This
concentration of F127 is commonly used in commercial
products for human consumption at concentrations of
5% or below. Oral LD50 in rats for F127 is >10 g kgG1 22
and at a 5% concentration, the daily dose of F127
ingested would be less than 10 g kgG1. RES solutions
were prepared fresh (1 L at a time) to obtain final
concentrations of 0.15 mg mLG1 (low dose group) or
0.3 mg mLG1 (high dose group). Considering the
average rat weight and average daily water intake, these
© 2015 Science Reuters, UK
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PHARMACOLOGIA
RESEARCH ARTICLE
transferred electrophoretically (30 min at 20V) to
PVFD membrane and incubated with
CYP4A1/CYP4A2/CYP4A3 rabbit anti-rat polyclonal
antibody, at a 1:1000 dilution (Lifespan Biosciences)
overnight at 4°C. After washes with PBST, the
secondary antibody (goat anti-rabbit conjugated to
horseradish peroxidase, Thermo Scientific) was added at
a 1:5000 dilution for 1 h at room temperature. The
immunoreactive proteins were detected via enhanced
chemiluminescence and x-ray film imaging and the
resultant signals were analyzed by densitometry
(Bio-Rad). The intensity of the band was normalized to
GADPH signals, which was used as loading control.
Tissue collection: At the end of the study, rats were
anesthetized with isoflurane, the abdominal cavities were
opened and the kidneys were rapidly removed and rinsed
with ice-cold saline. Kidney tissues were then flash
frozen in liquid nitrogen and stored at -80°C until use.
Preparation of microsomes and measurement of
protein content: Kidney microsomes were prepared as
described previously 25 . Microsomal protein
concentrations were determined in triplicate using
bovine serum albumin as a calibration standard as
described before26. Absorbance was measured at 750 nm
on a Synergy2® micro-plate reader using Gen5 Software
(BioTek, Winooski, VT).
Epoxide Hydrolase activity (sEH): Metabolism of
Epoxy Fluor 7 (Cayman Chemical Co.) to a fluorescent
metabolite was utilized to determine the sEH activity in
kidney microsomes as described previously29. Briefly,
reactions were carried out in mixtures (200 μL)
containing 25 mM Bis Tris-HCl, 1 mg mLG1 BSA,
Epoxy Fluor 7 and rat kidney microsomes (equivalent to
10 μg protein). The resulting solution was incubated at
37°C in a black 96-well flat bottom plate. The
fluorescence of the Epoxy Fluor 7 metabolite was
determined using an excitation wavelength of 330 nm
and emission wavelength of 465 nm on a Synergy2®
microplate reader using Gen5 Software (BioTek)
Microsomal Arachidonic Acid (AA) hydroxylation
and analysis of 20-HETE formation: Oxidation of
Arachidonic Acid (AA) to its metabolite 20-HETE was
determined in reaction mixtures (500 μL) containing
100 mM phosphate buffer (pH 7.4), 40 μM AA, 2 mM
MgCl2, 1 mM NADPH and rat kidney microsomes
equivalent to 0.4 mg protein. Reactions were initiated
with NADPH and were terminated after 15 min at 37°C
with 1.0 M HCl. In vitro experiments using renal cortical
microsomes from untreated Sprague-Dawley rats were
similarly utilized to study the effect of RES on 20-HETE
formation in vitro. In these experiments, microsomes
were exposed to different RES concentrations (1, 10 and
50 μM) in presence and absence of NADPH. 20-HETE
formation rate in both in vitro and in vivo studies was
measured as described previously27 with little
modifications. Briefly, the reaction mixtures were
extracted with ethyl acetate and the organic extracts were
evaporated with nitrogen gas and the residues were
reconstituted in the HPLC mobile phase. AA and
20-HETE were resolved on an Agilent Eclipse Plus C18
column (4.6×250 mm; Agilent Technologies, Santa
Clara, CA) with UV detection at 200 nm. Initial mobile
phase composition was 45% acetonitrile in water with
0.1% acetic acid. Linear gradient (0.5% minG1) was
utilized over 30 min, followed by a sudden increase to
20% minG1 to 100% acetonitrile. Flow rate was
maintained at 0.3 mL minG1 and column temperature at
40°C.
Data analysis: Data is reported as Mean±SD.
Differences in 20-HETE formation rate, CYP activity,
level of protein expression and differences in MAP
among groups, were assessed by one-way Analysis of
Variance (ANOVA) with Tukey’s post-hoc test for pairwise multiple comparisons. Correlation analysis was also
performed to investigate whether a change in blood
pressure corresponds to a change in CYP4A or sEH
activities. Statistical analysis was conducted using
GraphPad Prism 5.0 (GraphPad Software Inc., San
Diego, CA). Tukey post hoc test was used for pair-wise
comparison. The p<0.05 was considered as statistically
significant.
RESULTS
Exposure of rats to different doses of resveratrol
(RES) had no obvious adverse effect, such as behavioral
changes or loss of appetite and the gain in body weight
was not significantly different in treatment groups
compared to control animals.
Immunoquantification of CYP4A in rat kidney
microsomes: Rat kidney microsomes (10 kg per lane,)
were incubated at 100°C for 5 min in Laemmli
sample buffer (BioRad, Hercules, CA) and
electrophoresed for 1 h at constant voltage (150 V)
through precast polyacrylamide gels (BioRad) with
minor modifications28. Microsomal proteins were then
© 2015 Science Reuters, UK
Effect of resveratrol treatment on Mean Arterial
Pressure (MAP): To determine the effect of RES on
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Mean arterial pressure (mmHg)
RESEARCH ARTICLE
200
(a)
180
160
140
0
30
Changes in MAP
(mmHg) (week 7-0)
of the 7 week treatment with a net decrease of
7.2±19.2 mm Hg or 4.22% decrease from the baseline.
Although, both treatment groups showed MAP
reduction at the end of the 7 week treatment compared
to the control group, this reduction was only significant
in the high-dose RES treatment group(One-way
ANOVA p = 0.0017; Tuckey’s post-hoc test p<0.05 for
control-high RES).
Control
1
RES 20 mg kg¯
1
RES 40 mg kg¯
1
2
3
4
5
Treatment (weeks)
6
7
Effect of RES treatment on arachidonic acid
metabolism: Previous studies have shown that CYP4A
enzymes are entirely responsible for catalyzing the
ω-hydroxylation of (AA) to the potent vasoconstrictor
metabolite, 20-HETE in the rat kidney12. To examine if
the observed reduction in blood pressure in SHR rats
by RES could be associated with inhibition of
CYP4A-mediated formation of 20-HETE, this
reaction was measured in vitro using renal cortical
microsomes from untreated Sprague-Dawley rats.
Microsomes were pre-incubated with or without RES
(1, 10 and 50 μM) in the presence of NADPH before
adding AA (100 μM) and other incubation components.
An HPLC-UV method was utilized for the
quantification of 20-HETE in the incubation mixture
and measurement of 20-HETE formation rate as
previously described30. Under these chromatographic
conditions, 20-HETE and AA were eluted with retention
times of 36 and 38 min, respectively (Fig. 3b).
Additionally as shown in Fig. 3a, 20-HETE formation
was NADPH-dependent. This may indicate that
inhibition of 20-HETE formation by RES is
mechanism-based as NADPH was required in the
incubation mixture for inactivation of CYP enzymes.
The 20-HETE formation was inhibited by in vitro
inactivation with RES in renal cortical microsomes in a
concentration-dependent manner. At RES
concentrations of 10 and 50 μM, 20-HETE formation
rate was reduced to 75 and 67% of control (Fig. 4a,
in vitro).
The effect of the in-vivo administration of RES on
AA metabolism was measured in rat renal microsomes
after 7 weeks of exposure to RES in the drinking water
at concentrations of 20 and 40 mg kgG1. Similar to the
observed in vitro results, AA ω-hydroxylation was
inhibited by RES treatment, however this inhibition
was only significant at the high-dose of RES compared
to control rats, (Fig. 4b, in vivo).
(b)
20
10
0
-10
*
-20
Control
RES 20 mg kg¯1
RES 40 mg kg¯1
Treratment (week)
Fig. 2(a-b): Effect of RES on MAP in SHR rats. Male
14-week-old SHR rats (n = 5-7/group)
were exposed to RES in their drinking water
at concentrations of 0, 20 and 40 mg kgG1
for 7 weeks. MAP was measured noninvasively once every week. Change in MAP
is expressed as Mean±SD of 5 (REStreated) or 7(control) animals. (a) Changes
in MAP in SHR over 7 weeks and (b)
Difference in MAP between the end and the
beginning of the treatment, i.e., (MAP at
week 7)-(MAP at week 0). *Significant
difference from control with p<0.05
(95% CI: 5.505-50.17)
blood pressure in SHR rats, the MAP of all groups was
measured once weekly. Figure 2a shows the MAP in rats
during baseline conditions and after exposure to low
dose (20 mg kgG1) and high dose (40 mg kgG1) of RES.
During the study, control rats showed a progressive rise
in their MAP from 166.3±1.9 to 185.4±10.8 mm Hg
after 7 weeks, an average increase of 20.7±9.1 mm Hg
that represents 12.45% increase compared to baseline. In
contrast, exposure of SHR rats to even the low dose of
RES prevented the expected progressive rise in MAP
with a net decrease of 2.6±15.7 mm Hg that represents
1.55% decrease compared to baseline. Moreover,
administration of RES in the high dose resulted in a
more substantial decrease in MAP from 166.8±2.2 at
the beginning of the study to 159.6±17.7 at the end
© 2015 Science Reuters, UK
Effect of RES treatment on CYP4A protein levels:
To examine the effect of RES on the expression level of
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PHARMACOLOGIA
RESEARCH ARTICLE
Absorbance at (200 nm) (mAU)
(a)
(a)
1050
1000
950
900
850
800
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
0
AA
5.0
800
10.0
15.0
20.0
25.0
30.0
35.0
40.0
(b)
Absorbance at (200 nm) (mAU)
700
600
20-HETE
500
400
300
AA
200
100
0
-100
-200
-300
-400
-500
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
35.0
37.5
40.0
42.5
45.0
min
Time (min)
Fig. 3(a-b): HPLC analysis of AA metabolism by rat kidney microsomes representative chromatogram produced after
incubation of AA with, (a) Untreated Sprague-Dawley rat kidney microsomes in the absence of NADPH
and (b) Kidney microsomes from SHRs treated with 20 mg kgG1 RES in drinking water for 7 weeks
CYP4A protein, western blotting of renal microsomes
was performed using a polyclonal antibody against rat
CYP4A1/2/3. As shown in Fig. 5, RES-mediated
inhibition of AA ω-hydroxylation was associated
with a loss of CYP4A immunoreactive protein.
Similar to the observed dose-dependent inhibition of
AA hydroxylase activity, RES also caused a significant
inhibition of CYP4A protein expression compared to
the control group but no significant difference was
observed between the two RES treatment groups
(Fig. 5).
different concentrations of RES were utilized as
explained briefly in the “Materials and Methods”
section29. Although the low dose RES did not have
any effect on sEH, the high dose RES significantly
inhibited the activity to 73% of the control value
(Fig. 6).
As establishing a cause-effect relationship between
the observed effect of RES on MAP, CYP4A and
sEH activity was not possible with the presented
study, the correlation between these variables was
investigated. When RES-dependent changes in MAP
were compared to changes in 20-HETE formation
rate as a measure of CYP4A activity, a good correlation
was observed (Pearson r; -0.85 and p: 0.038). While
20 and 40 mg kgG1 RES doses reduced MAP by
approximately 2 and 9%, 20-HETE formation rate was
reduced by 11 and 36%, respectively (Fig. 7).
Similarly, changes in MAP reasonably correlate
(Pearson r; -0.75 and p: 0.046) with changes in sEH
Effect of RES treatment on soluble epoxide
hydrolase activity: Several studies have provided
compelling evidence that sEH plays a critical role in the
metabolic conversion of the anti-hypertensive
eicosanoids to inactive metabolites. To further study the
effect of RES treatment on sEH activity, kidney
microsomes from control rats or rats exposed to
© 2015 Science Reuters, UK
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PHARMACOLOGIA
150
(a)
20-HETE formation rate (control %)
20-HETE formation rate (control %)
RESEARCH ARTICLE
100
*
*
50
0
0
10
1
RES concentration (µM)
150 (b)
100
50
0
Control
50
RES 20 mg kg¯
1
RES 40 mg kg¯
1
Fig. 4(a-b): Inhibition of AA ω-hydroxylation activity by RES both in vitro and in vivo (a) Renal cortical microsomes
from untreated Sprague Dawley rats were incubated with various concentrations of RES in the presence
of NADPH. RES-treated microsomes were then used to measure 20-HETE formation. Values, average
of 5 replicates, are expressed as % of control (RES-free microsomes) and (b) Male SHR rats (n = 5/
group) were administered 20 or 40 mg kgG1 RES in their drinking water for 7 weeks and kidneys were
harvested at the end of the study. The NADPH-dependent formation of 20-HETE was measured in renal
cortical microsomes. Values are expressed as % of control (rats that received RES-free water, n = 7). All
groups were compared using one-way ANOVA followed by multiple comparisons. *Significant difference
from control with p<0.05
Relative kidney CYP4A
abundance (control %)
200
(a)
150
100
*
50
0
RES 20 mg kg¯1
Group
Control
1
2
3
4
5
6
7
8
9
RES 40 mg kg¯1
10
11
12
13
14
C YP 4A
GAPDH
Control
R ES 20 mg kg-1
RES 40 mg kgA-1
Fig. 5(a-b): Effect of RES on kidney expression of CYP4A. (a) Renal CYP4A protein levels in rats (n = 5-7/group)
treated for 7 weeks with RES at 20 and 40 mg kgG1, expressed as the percentage of control levels in
untreated rats. The immunoreactive proteins were detected via enhanced chemiluminescence and x-ray
film imaging and the resultant signals were analyzed by densitometry. The intensity of the band was
normalized to a loading control (GAPDH signals), then data was expressed as percent of control and (b)
Original specimen analyzed by densitometry (Control; Lanes 1-4, Low RES dose: Lanes 5-9, High RES
dose: Lanes 10-14). *Significant difference from control with p<0.05
activity in a RES dose-dependent manner. Namely,
compared to 2 and 9% reduction in MAP, sEH activity
© 2015 Science Reuters, UK
was reduced by 5 and 25% following administration of
20 and 40 mg kgG1 RES, respectively (Fig. 7).
366
150
20
Change in MAP (%)
125
100
*
75
50
25
100
10
5
75
0
0
-5
20
40
-10
50
25
-15
-20
0
Control
RES 20 mg kg¯1
RES 40 mg kg¯1
20
Fig. 6: Effect of RES on activity of kidney sEH Mean
epoxide hydrolase (sEH) activity in rats
(n = 5/RES and n = 7/control groups) treated
for 7 weeks with RES at 20, 40 mg kgG1,
expressed as the percentage of control
fluorescence in rats that received RES-free
water. Rat kidney microsomes (equivalent to
10 μg protein) were incubated with Epoxy Fluor
7 and the fluorescence of the Epoxy Fluor 7
metabolite was determined. Data is presented
as Mean±SD. *Significant difference with
p<0.05
120
(b)
15
Change in MAP (%)
110
10
5
100
0
-5
0
20
-10
40
90
80
-15
-20
RES dose (mg kg¯1)
70
Fig. 7(a-b): Correlation of changes in MAP, CYP
activity and sEH activity as a function of
RES dose. Percent Changes in MAP
(closed circles) in correlation to (a) CYP4A
activity (open squares and (b) sEH activity
(closed squares) as a function of RES dose.
MAP is presented as % change from
baseline (week 0). Both CYP4A and sEH
activities are presented as a % control
activity. While treatment rats received RES
in their drinking water in doses of 20 or
40 mg kgG1 for 7 weeks, control rats did not
receive RES
DISCUSSION
As previously demonstrated that sulforaphane, the
main active isothiocyante in cruciferous vegetable,
reduced the progressive rise in blood pressure in
Spontaneously Hypertensive Rats (SHR) through
modulation of Arachidonic Acid (AA) metabolism30.
This study was undertaken to investigate possible
mechanisms that contribute to the beneficial effect of
resveratrol (RES) on blood pressure in hypertensive rats.
The results show for the first time that treatment of
SHR with RES significantly prevents the progressive rise
in blood pressure, reduces the expression and activity of
renal CYP4A isozymes responsible for 20-HETE
formation and reduces the activity of soluble epoxide
hydrolase (sEH) responsible for the degradation of the
vasodilator metabolites EETs.
RES is a naturally-occurring compound that exhibits
cytoprotective, antioxidant and anti-inflammatory
vasoprotective properties31. Most studies examining the
mechanism of action of RES proposed that preventing
and/or inhibiting the development of cardiovascular
diseases is related to its ability to reduce oxidative stress
and associated inflammation and thus, ameliorate
diseases that become more common with aging, such as
hypertension23,31. For example, the consumption of
25 mg kgG1 RES in drinking water for 14 days reduced
© 2015 Science Reuters, UK
0
(EH activity control %)
Relative kidney CYP4A
abundance (control %)
sHE activity
Percentage changes in MAP
CYP4A activity
(a)
15
CYP4A activity control %)
PHARMACOLOGIA
RESEARCH ARTICLE
oxidative stress, improved endothelial functions and
protected rats against development of pulmonary
hypertension23. Furthermore, consumption of diets
containing high dose RES by SHRs was found to
improve vascular function, attenuate high BP and
prevent cardiac hypertrophy.
Whether this anti-hypertensive and cardioprotective
effect of RES is due to antioxidant effect is not yet
established. Additionally, if the anti-hypertensive effects
of RES are due to exclusively because of its antioxidant
properties, then other antioxidants should also show
similar anti-hypertensive properties. However, results of
three randomized clinical trials testing the effects of the
antioxidant vitamin E on hypertension have proven
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disappointing32-34. Similarly, a non-significant reduction
in blood pressure was reported in vitamin D-treated
patients35. Accordingly, it is conceivable that RES
reduces blood pressure by more than one mechanism.
This study is the first to investigate the effect of RES
on modulation of AA-metabolizing enzymes in the
kidney, as a potential mechanism for its antihypertensive property. Even though AA and its
metabolites are present in numerous organs, including
the liver, lungs and brain, the kidney plays a central role
in arterial blood pressure regulation through both local
and hormonal mechanisms. The AA metabolites, such as
20-HETE, are known to have a strong vasoconstrictive
effect on the renal afferent arteriole that, in turn, is likely
to trigger the powerful Renin-Angiotensin-Aldosterone
(RAA) cascade, one of primary targets of
anti-hypertensive therapy. The fact that 20-HETE also
has a direct inhibitory effect on sodium reabsorption
which is opposite to the RAA-mediated sodium
retention, is likely to explain the less pronounced
changes in blood pressure during a global downregulation of 20-HETE formation, as in the current
study.
To avoid inducing any stress-related effect on blood
pressure and also to simulate the commonly used route
of RES administration was introduction RES to rats in
their drinking water rather than oral gavage or
subcutaneous injection. Stability studies using
absorbance measurements indicate that RES is stable up
to 0.25 mg mLG1 in 5% Pluronic® F127 solutions at
room temperature for 3 days. Notably, the observed
attenuation of the expected rise in blood pressure by
RES after a 7 week period was comparable to the results
achieved with commonly used anti-hypertensive
medication in the same rat model. For example,
Telmisartan (5 mg for 8 weeks), Indapamide (1 mg kgG1
for 8 weeks), Hydrochlorothiazide (20 mg kgG1 for 8
weeks) and Metoprolol (100 mg kgG1) with Hydralazine
(50 mg kgG1 for 10 weeks) resulted in an average MAP
reduction of 25, 17, 15 and 22 mm Hg; respectively36,37.
Such reduction in blood pressure was associated with a
similar pattern of inhibition in the formation rate of
20-HETE in kidney microsomes. It is well established
that 20-HETE induces hypertension through its potent
vasoconstriction in the renal and peripheral vasculature
and by promoting sodium retention3. Considerable
evidence has now accumulated suggesting that
development of hypertension in the SHR strain is, at
least in part, attributed to the elevated production of
20-HETE in the kidney and mesenteric arteries12. The
presented data are consistent with this hypothesis and
with several findings that inhibitors of 20-HETE
synthesis lower blood pressure by 10-15 mm Hg in
© 2015 Science Reuters, UK
SHR13. In attempts to identify the cytochrome P450
(CYP) isoforms responsible for the renal metabolism of
AA and formation of 20-HETE using selective inhibitors
and anti-sense oligonucleotides, it has been identified
that CYP4A1 exhibits the highest catalytic activity for the
formation of 20-HETE in the rat kidney, followed by
CYP4A2 and CYP4A37,38. Using specific antibody against
CYP4A1/2/3, the immunoblotting data indicate that RES
lowers the expression of these isoforms in a manner
similar to that involved in reducing MAP and 20-HETE
formation.
It has also been established that EET metabolites of
AA produced by the endothelium are potent vasodilators
and natriuretic agents and are proposed as endotheliumderived hyperpolarizing factors39. Degradation of these
metabolites by sEH led to more research interest towards
inhibiting this enzyme as an avenue to increase EETs
levels and prevent progression of hypertension40. In
addition to elevated renal production of 20-HETE,
SHRs also exhibit an increased expression of renal sEH
compared to their normotensive WKY control rats41,42.
Specific inhibitors to sEH enzyme lowered blood
pressure in SHR, but not in WKY rats41. Such findings
may indicate that sEH contributes to the initiation of
mechanisms that eventually result in high blood pressure
in SHR. The presented data indicate for the first time
that both low and high RES doses inhibit the activity of
sEH in the kidney of SHR and this inhibition was
parallel to the observed reduction in blood pressure.
CONCLUSION
The presented results have shown that even the
lower RES dose prevented the progressive rise in MAP
while the higher RES dose significantly reduced MAP in
hypertensive rats. For the first time, it was demonstrated
that the lowering of blood pressure was accompanied by
modulation of AA metabolism through inhibiting both,
the formation of 20-HETE and activity of sEH. Those
results contribute the novel knowledge to the antihypertensive properties of RES.
Future studies will be undertaken to examine
whether an earlier exposure to RES can prevent the
development of hypertension and whether RES can
reverse the established hypertension in older SHR rats.
Additionally, the effect of specific inhibitors or inducers
for the 20-HETE formation pathway will be examined
on the observed anti-hypertensive effect of RES in SHR
rats.
ACKNOWLEDGMENTS
We thank Bryan Bustamante for assistance in
measuring blood pressure and Anik Amin, Margarita
Can and Tamara Olenyik for microsomal preparation,
368
PHARMACOLOGIA
RESEARCH ARTICLE
HPLC and immunoblotting assays. This study was
supported in part by the Medical Research Foundation
of Oregon (MRF), Collins Medical Trust and Faculty
Development Grant from Pacific University, Oregon
(F.E.).
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Physiol., 275: R426-R438.
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